RESUMO
Filamentous bacteriophages belonging to the order Tubulavirales, family Inoviridae, significantly affect the properties of Gram-negative bacteria, but filamentous phages of many important pathogens have not been described so far. The aim of this study was to examine A. baumannii filamentous phages for the first time and to determine their effect on bacterial virulence. The filamentous phages were detected in 15.3% of A. baumannii strains as individual prophages in the genome or as tandem repeats, and a slightly higher percentage was detected in the culture collection (23.8%). The phylogenetic analyses revealed 12 new genera within the Inoviridae family. Bacteriophages that were selected and isolated showed structural and genomic characteristics of the family and were unable to form plaques. Upon host infection, these phages did not significantly affect bacterial twitching motility and capsule production but significantly affected growth kinetics, reduced biofilm formation, and increased antibiotic sensitivity. One of the possible mechanisms of reduced resistance to antibiotics is the observed decreased expression of efflux pumps after infection with filamentous phages.
Assuntos
Acinetobacter baumannii , Biofilmes , Genoma Viral , Filogenia , Acinetobacter baumannii/virologia , Acinetobacter baumannii/genética , Biofilmes/crescimento & desenvolvimento , Inovirus/genética , Inovirus/fisiologia , Inovirus/isolamento & purificação , Especificidade de Hospedeiro , Antibacterianos/farmacologia , Virulência , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Bacteriófagos/classificação , Prófagos/genética , Prófagos/fisiologiaRESUMO
Many filamentous vibriophages encode virulence genes that lead to the emergence of pathogenic bacteria. Most genomes of filamentous vibriophages characterized up until today were isolated from human pathogens. Despite genome-based predictions that environmental Vibrios also contain filamentous phages that contribute to bacterial virulence, empirical evidence is scarce. This study aimed to characterize the bacteriophages of a marine pathogen, Vibrio alginolyticus (Kiel-alginolyticus ecotype) and to determine their role in bacterial virulence. To do so, we sequenced the phage-containing supernatant of eight different V. alginolyticus strains, characterized the phages therein and performed infection experiments on juvenile pipefish to assess their contribution to bacterial virulence. We were able to identify two actively replicating filamentous phages. Unique to this study was that all eight bacteria of the Kiel-alginolyticus ecotype have identical bacteriophages, supporting our previously established theory of a clonal expansion of the Kiel-alginolyticus ecotype. We further found that in one of the two filamentous phages, two phage-morphogenesis proteins (Zot and Ace) share high sequence similarity with putative toxins encoded on the Vibrio cholerae phage CTXΦ. The coverage of this filamentous phage correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phage contributes to bacterial virulence.
Assuntos
Caudovirales/genética , Genoma Bacteriano , Inovirus/genética , Vibrio alginolyticus/genética , Vibrio alginolyticus/virologia , Animais , Carga Bacteriana , Caudovirales/classificação , Caudovirales/isolamento & purificação , DNA Viral , Doenças dos Peixes/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Inovirus/classificação , Inovirus/isolamento & purificação , Vibrioses/veterinária , Vibrio alginolyticus/classificação , Vibrio alginolyticus/patogenicidade , VirulênciaRESUMO
A filamentous bacteriophage, designated ÏRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ÏRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ÏRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ÏRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ÏRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ÏRSM phages, ÏRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ÏRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ÏRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ÏRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.
Assuntos
Genoma Viral , Inovirus/genética , Inovirus/isolamento & purificação , Doenças das Plantas/virologia , Ralstonia solanacearum/virologia , Virulência/genética , DNA Viral/genética , Índia , Inovirus/metabolismo , Filogenia , Prófagos/genética , Ralstonia solanacearum/crescimento & desenvolvimento , Ralstonia solanacearum/patogenicidadeRESUMO
Unlike Ff-like coliphages, certain filamentous Inoviridae phages integrate their genomes into the host chromosome and enter a prophage state in their infectious cycle. This lysogenic life cycle was first reported for Xanthomonas citri Cf phage. However, except for the X. citri phages Cf and XacF1, complete genome sequence information about lysogenic Xanthomonas phages is not available to date. A proviral sequence of Xf109 phage was identified in the genome of Xanthomonas oryzae, the rice bacterial blight pathogen, and revived as infectious virions to lysogenize its host de novo. The genome of Xf109 phage is 7190 nucleotides in size and contains 12 predicted open reading frames in an organization similar to that of the Cf phage genome. Seven of the Xf109 proteins show significant sequence similarity to Cf and XacF1 phage proteins, while its ORF4 shares 92 % identity with the major coat protein of X. phage oryzae Xf. Integration of Xf109 phage DNA into the host genome is site-specific, and the attP/attB sequence contains the dif core sequence 5'-TATACATTATGCGAA-3', which is identical to that of Cf, XacF1, and Xanthomonas campestris phage ÏLf. To my knowledge, this is the first complete genome sequence of a filamentous bacteriophage that infects X. oryzae.
Assuntos
Cromossomos Bacterianos/química , DNA Viral/genética , Genoma Viral , Inovirus/genética , Mutagênese Insercional , Xanthomonas/virologia , Sequência de Bases , Proteínas do Capsídeo/genética , Mapeamento Cromossômico , Expressão Gênica , Inovirus/isolamento & purificação , Lisogenia , Fases de Leitura Aberta , Oryza/microbiologia , Alinhamento de Sequência , Vírion/genéticaRESUMO
Bacterial genomes commonly contain prophage sequences as a result of past infections with lysogenic phages. Many of these integrated viral sequences are believed to be cryptic, but prophage genes are sometimes coopted by the host, and some prophages may be reactivated to form infectious particles when cells are stressed or mutate. We found that a previously uncharacterized filamentous phage emerged from the genome of Acinetobacter baylyi ADP1 during a laboratory evolution experiment. This phage has a genetic organization similar to that of the Vibrio cholerae CTXÏ phage. The emergence of the ADP1 phage was associated with the evolution of reduced transformability in our experimental populations, so we named it the competence-reducing acinetobacter phage (CRAÏ). Knocking out ADP1 genes required for competence leads to resistance to CRAÏ infection. Although filamentous bacteriophages are known to target type IV pili, this is the first report of a phage that apparently uses a competence pilus as a receptor. A. baylyi may be especially susceptible to this route of infection because every cell is competent during normal growth, whereas competence is induced only under certain environmental conditions or in a subpopulation of cells in other bacterial species. It is possible that CRAÏ-like phages restrict horizontal gene transfer in nature by inhibiting the growth of naturally transformable strains. We also found that prophages with homology to CRAÏ exist in several strains of Acinetobacter baumannii These CRAÏ-like A. baumannii prophages encode toxins similar to CTXÏ that might contribute to the virulence of this opportunistic multidrug-resistant pathogen. IMPORTANCE: We observed the emergence of a novel filamentous phage (CRAÏ) from the genome of Acinetobacter baylyi ADP1 during a long-term laboratory evolution experiment. CRAÏ is the first bacteriophage reported to require the molecular machinery involved in the uptake of environmental DNA for infection. Reactivation and evolution of CRAÏ reduced the potential for horizontal transfer of genes via natural transformation in our experiment. Risk of infection by similar phages may limit the expression and maintenance of bacterial competence in nature. The closest studied relative of CRAÏ is the Vibrio cholerae CTXÏ phage. Variants of CRAÏ are found in the genomes of Acinetobacter baumannii strains, and it is possible that phage-encoded toxins contribute to the virulence of this opportunistic multidrug-resistant pathogen.
Assuntos
Acinetobacter baumannii/virologia , Inovirus/isolamento & purificação , Prófagos/isolamento & purificação , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Genoma Viral , Inovirus/classificação , Inovirus/genética , Inovirus/fisiologia , Prófagos/classificação , Prófagos/genética , Prófagos/fisiologiaRESUMO
A low-temperature-inducible protein expression vector (pSW2) based on a filamentous phage (SW1) of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. This vector replicated stably in Escherichia coli and Shewanella species, and its copy number increased at low temperatures. The pSW2 vector can be utilized as a complementation plasmid in WP3, and it can also be used for the production of complex cytochromes with multiple heme groups, which has the potential for application for metal ion recovery or bioremediation. Promoters of low-temperature-inducible genes in WP3 were fused into the vector to construct a series of vectors for enhancing protein expression at low temperature. The maximum green fluorescent protein intensity was obtained when the promoter for the hfq gene was used. The WP3/pSW2 system can efficiently produce a patatin-like protein (PLP) from a metagenomic library that tends to form inclusion bodies in E. coli. The yields of PLP in the soluble fraction were 8.3 mg/liter and 4.7 mg/liter of culture at 4°C and 20°C, respectively. Moreover, the pSW2 vector can be broadly utilized in other Shewanella species, such as S. oneidensis and S. psychrophila.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Vetores Genéticos , Inovirus/genética , Plasmídeos , Shewanella/genética , Shewanella/virologia , Fusão Gênica Artificial , Replicação do DNA , Genes Reporter , Genética Microbiana/métodos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Inovirus/isolamento & purificação , Biologia Molecular/métodos , Regiões Promotoras Genéticas , Recombinação Genética , TemperaturaRESUMO
Filamentous bacteriophage RS611 (ÏRS611), which infects the phytopathogen Ralstonia solanacearum, had a circular single-stranded DNA genome that was characterized as an Ff-type phage belonging to the family Inoviridae. The ÏRS611 genome was composed of 6386 bases with a G + C content of 62.1 % and contained 11 putative open reading frames. The ÏRS611 genome showed high similarity to those of Ralstonia phages RSS0 and RSS1. However, approximately 900-nucleotide deletions were found in the region corresponding to open reading frames 10 and 11 of ÏRSS0 and ÏRSS1.
Assuntos
Vírus de DNA/genética , DNA Viral/química , DNA Viral/genética , Genoma Viral , Inoviridae/genética , Inovirus/genética , Ralstonia solanacearum/virologia , Composição de Bases , Vírus de DNA/isolamento & purificação , DNA Circular/genética , Inoviridae/classificação , Inoviridae/isolamento & purificação , Inovirus/classificação , Inovirus/isolamento & purificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência , SinteniaRESUMO
A filamentous bacteriophage (Ï), ÏRS603, which is infectious to the phytopathogen Ralstonia solanacearum was isolated. ÏRS603 was found to have a circular single-stranded DNA genome composed of 7679 nucleotides and to contain 13 putative open reading frames (ORFs). The ÏRS603 genome showed strong similarity with those of Ralstonia phages ÏRSM1 and ÏRSM3, as reported by Askora et al. The ÏRS603 genome had no ORFs corresponding to ORFs 2, 3, 13 and 14 (integrase) of ÏRSM3. ÏRS603 had an ORF that was homologous to other Ralstonia phages ÏRSS0 and ÏRSS1; however, ÏRSM1 and ÏRSM3 did not.
Assuntos
DNA de Cadeia Simples/genética , DNA Viral/genética , Genoma Viral , Inovirus/genética , Ralstonia solanacearum/virologia , Proteínas Virais/genética , Sequência de Aminoácidos , DNA de Cadeia Simples/química , DNA Viral/química , Inovirus/química , Inovirus/isolamento & purificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/químicaRESUMO
CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ.
Assuntos
Vetores Genéticos , Genética Microbiana/métodos , Inovirus/genética , Biologia Molecular/métodos , Vibrio/virologia , Sítios de Ligação Microbiológicos , Cromossomos Bacterianos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Escherichia coli/genética , Expressão Gênica , Genoma Viral , Inovirus/isolamento & purificação , Klebsiella pneumoniae/genética , Regiões Promotoras Genéticas , Recombinação Genética , Salmonella enterica/genéticaRESUMO
Two novel filamentous phages, phiSMA6 and phiSMA7, were isolated from Stenotrophomonas maltophilia environmental strain Khak84. We identified and annotated 11 potential open reading frames in each phage. While the overall layout of the functional gene groups of both phages was similar to that of the known filamentous phages, they differed from them in their molecular structure. The genome of phiSMA6 is a mosaic that evolved by acquiring genes from at least three different filamentous S. maltophilia phages and one Xanthomonas campestris phage related to Cf1. In the phiSMA6 genome, a gene similar to the bacterial gene encoding the mating pair formation protein trbP was also found. We showed that phiSMA6 possesses lysogenic properties and upon induction produces high-titer lysates. The genome of phiSMA7 possesses a unique structure and was found to be closely related to a prophage present in the chromosome of the completely sequenced S. maltophilia clinical strain D457. We suggest that the other three filamentous phages of S. maltophilia described previously also have the capacity to integrate into the genome of their bacterial host.
Assuntos
Inovirus/genética , Inovirus/fisiologia , Stenotrophomonas maltophilia/virologia , Integração Viral , DNA Viral/química , DNA Viral/genética , Genes Virais , Inovirus/isolamento & purificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Recombinação Genética , Análise de Sequência de DNARESUMO
Low-temperature ecosystems represent the largest biosphere on Earth, and yet our understanding of the roles of bacteriophages in these systems is limited. Here, the influence of the cold-active filamentous phage SW1 on the phenotype and gene transcription of its host, Shewanella piezotolerans WP3 (WP3), was investigated by construction of a phage-free strain (WP3ΔSW1), which was compared with the wild-type strain. The expression of 49 genes, including 16 lateral flagellar genes, was found to be significantly influenced by SW1 at 4°C, as demonstrated by comparative whole-genome microarray analysis. WP3ΔSW1 was shown to have a higher production of lateral flagella than WP3 and enhanced swarming motility when cultivated on solid agar plates. Besides, SW1 has a remarkable impact on the expression of a variety of host genes in liquid culture, particularly the genes related to the membrane and to the production of lateral flagella. These results suggest that the deep-sea bacterium WP3 might balance the high-energy demands of phage maintenance and swarming motility at low temperatures. The phage SW1 is shown to have a significant influence on the swarming ability of the host and thus may play an important role in adjusting the fitness of the cells in the deep-sea environment.
Assuntos
Temperatura Baixa , Flagelos/metabolismo , Inovirus/fisiologia , Shewanella/genética , Genes Bacterianos , Processamento de Imagem Assistida por Computador , Inovirus/isolamento & purificação , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/genética , Shewanella/crescimento & desenvolvimento , Shewanella/virologiaRESUMO
Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJΦ, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJΦ is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which are organized in a modular structure. VFJΦ was found to be a mosaic of two groups of V. cholerae phages. A large part of the genome is highly similar to that of the fs2 phage, and the remaining 700 bp is homologous to VEJ and VCYΦ. This 700 bp region gave VFJΦ several characteristics that are not found in fs2 and other filamentous phages. In its native host ICDC-4470 and newly-infected strain N16961, VFJΦ was found to exist as a plasmid but did not integrate into the host chromosome. It showed a relatively wide host range but did not infect the classical biotype O1 V. cholerae strains. After infection, the host strains exhibited obvious inhibition of both growth and flagellum formation and had acquired a low level of ampicillin resistance and a high level of kanamycin resistance. The antibiotic resistances were not directly conferred to the hosts by phage-encoded genes and were not related to penicillinase. The discovery of VFJΦ updates our understanding of filamentous phages as well as the evolution and classification of V. cholerae filamentous phage, and the study provides new information on the interaction between phages and their host bacteria.
Assuntos
Inovirus/isolamento & purificação , Inovirus/fisiologia , Vibrio cholerae/virologia , Sequência de Bases , Genômica , Inovirus/genética , Fases de Leitura Aberta/genética , Plasmídeos/genética , Especificidade da Espécie , Integração Viral , Replicação ViralRESUMO
We report an ion exchange chromatographic purification method powerful for preparation of virus particles with ultrapure quality. The technology is based on large pore size monolithic anion exchangers, quaternary amine (QA) and diethyl aminoethyl (DEAE). These were applied to membrane-containing icosahedral bacteriophage PRD1, which bound specifically to both matrices. Virus particles eluted from the columns retained their infectivity, and were homogenous with high specific infectivity. The yields of infectious particles were up to 80%. Purified particles were recovered at high concentrations, approximately 5mg/ml, sufficient for virological, biochemical and structural analyses. We also tested the applicability of the monolithic anion exchange purification on a filamentous bacteriophage phi05_2302. Monolithic ion exchange chromatography is easily scalable and can be combined with other preparative virus purification methods.
Assuntos
Bacteriófago PRD1/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Inovirus/isolamento & purificação , Virologia/métodos , Bacteriófago PRD1/fisiologia , Inovirus/fisiologia , Viabilidade MicrobianaRESUMO
In this study, a novel filamentous phage, φSHP1, of the environmental Stenotrophomonas maltophilia strain P2 was isolated and characterized. Electron microscopy showed that φSHP1 resembled members of the family Inoviridae and was about 2.1 µm long. The 6,867-nucleotide genome of φSHP1 was a circular single-stranded DNA and had a replication form designated pSH1. Ten putative open reading frames (ORFs) were found in the φSHP1 genome, and six predicted proteins showed similarity to proteins in databases. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis of φSHP1 displayed one major structural polypeptide of approximately 4.0 kDa. N-terminal sequencing showed that it was the mature product of ORF5 and that its N-terminal 27 amino acid residues had been cleaved off from the predicted nascent protein. Finally, phylogenetic trees were constructed to analyze the phylogenetic relationship of φSHP1 to other known filamentous phages. φSHP1 appears to be the first reported Stenotrophomonas filamentous phage.
Assuntos
Inoviridae/classificação , Inoviridae/isolamento & purificação , Inovirus/classificação , Inovirus/isolamento & purificação , Stenotrophomonas maltophilia/virologia , Análise por Conglomerados , DNA Circular/genética , DNA Viral/química , DNA Viral/genética , Eletroforese em Gel de Poliacrilamida , Inoviridae/genética , Inoviridae/ultraestrutura , Inovirus/genética , Inovirus/ultraestrutura , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA , Proteínas Virais/química , Proteínas Virais/isolamento & purificaçãoRESUMO
Filamentous bacteriophages and their derivatives are showing great promise as a whole new class of industrial agents, such as biologically based nano-materials and viral vectors. This raises challenges for their large-scale manufacture, principally due to the lack of bioprocessing knowledge. This article addresses what will be a potentially important option in the primary purification of the bacteriophages. Polyethylene glycol (PEG)-salt dual precipitants, calcium ions, spermidine, and isoelectric precipitation were first examined for their potential suitability for bacteriophage concentration under both pure and broth conditions. Successful precipitants were further studied on the basis of their selective purification ability from DNA and protein contaminants in a clarified broth system. Both PEG-based and isoelectric precipitations resulted in bacteriophage purity improvements, and PEG-based precipitations offered the highest selectivities. This work shows that precipitation of bacteriophages can be an effective primary purification step in a large-scale bioprocess.
Assuntos
Escherichia coli/virologia , Microbiologia Industrial/métodos , Inovirus/isolamento & purificação , Inovirus/metabolismo , Fenômenos Bioquímicos , Cálcio/metabolismo , Precipitação Química , Meios de Cultura/metabolismo , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Nanoestruturas/química , Polietilenoglicóis/metabolismo , Proteínas/metabolismo , Espermidina/metabolismoRESUMO
Environmental Vibrio cholerae strains isolated from a coastal brackish pond (Oyster Pond, Woods Hole, MA) carried a novel filamentous phage, VCY, which can exist as a host genome integrative form (IF) and a plasmid-like replicative form (RF). Outside the cell, the phage displays a morphology typical of Inovirus, with filamentous particles â¼1.8 µm in length and 7 nm in width. Four independent RF isolates had identical genomes, except for 8 single nucleotide polymorphisms clustered in two regions. The overall genome size is 7,103 bp with 11 putative open reading frames organized into three functional modules (replication, structure and assembly, and regulation). VCY shares sequence similarity with other filamentous phages (including cholera disease-associated CTX) in a highly mosaic manner, indicating evolution by horizontal gene transfer and recombination. VCY integrates in the vicinity of the putative translation initiation factor Sui1 in chromosome II of V. cholerae. A screen of 531 closely related host isolates showed that â¼40% harbored phages, with 27% and 13% carrying the IF and RF, respectively. The relative frequencies of the RF and IF differed among strains isolated from the pond or lagoon of Oyster Pond, suggesting that the host habitat influences intracellular phage biology. The overall high prevalence within the host population shows that filamentous phages can be an important component of the environmental biology of V. cholerae.
Assuntos
Inovirus/isolamento & purificação , Lagoas/microbiologia , Vibrio cholerae/virologia , Microbiologia da Água , Sequência de Bases , DNA Bacteriano/isolamento & purificação , DNA Viral/isolamento & purificação , Genoma Viral , Inovirus/genética , Inovirus/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo ÚnicoRESUMO
AIMS: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. METHODS AND RESULTS: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single-strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. CONCLUSIONS: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single-strand DNA genome with an ORF encoding Zot-like protein. SIGNIFICANCE AND IMPACT OF THE STUDY: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.
Assuntos
Inovirus/genética , Ralstonia solanacearum/virologia , Sequência de Aminoácidos , DNA de Cadeia Simples/química , DNA Viral/química , Genoma Viral , Especificidade de Hospedeiro , Inovirus/isolamento & purificação , Inovirus/ultraestrutura , Solanum lycopersicum/microbiologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Rizosfera , Homologia de Sequência de Aminoácidos , Proteínas Virais/químicaRESUMO
A novel filamentous bacteriophage, designated VEJphi, was isolated from strain MO45 of Vibrio cholerae of the O139 serogroup. A molecular characterization of the phage was carried out, which included sequencing of its whole genome, study of the genomic structure, identification of the phage receptor, and determination of the function of some of the genes, such as those encoding the major capsid protein and the single-stranded DNA-binding protein. The genome nucleotide sequence of VEJphi, which consists of 6842 bp, revealed that it is organized in modules of functionally related genes in an array that is characteristic of the genus Inovirus (filamentous phages). VEJphi is closely related to other previously described filamentous phages of V. cholerae, including VGJphi, VSK and fs1. Like these phages, VEJphi uses as a cellular receptor the type IV fimbria called the mannose-sensitive haemagglutinin (MSHA). It was also demonstrated that VEJphi, like phage VGJphi, is able to transmit the genome of phage CTXphi, and therefore the genes encoding the cholera toxin (CT), horizontally among populations of V. cholerae expressing the MSHA receptor fimbria. This suggests that the variety of phages implicated in the horizontal transmission of the CT genes could be more diverse than formerly thought.
Assuntos
Toxina da Cólera/genética , Genoma Viral , Inovirus/genética , Vibrio cholerae/virologia , Proteínas de Fímbrias/metabolismo , Transferência Genética Horizontal , Inovirus/isolamento & purificação , Inovirus/metabolismo , Dados de Sequência Molecular , Receptores Virais/metabolismo , Análise de Sequência de DNA , Transdução Genética , Vibrio cholerae/genéticaRESUMO
Filamentous phage such as M13 and fd consist of a circular, single-stranded DNA molecule surrounded by several different coat proteins. These phages have been used extensively as vectors in phage display where one of the phage coat proteins is genetically engineered to contain a unique peptide surface loop. Through these peptide sequences, a phage collection can be screened for individual phage that binds to different macromolecules or small organic and inorganic molecules. Here, we use computer-controlled bioreactors to produce large quantities of filamentous phage in the bacterial host Escherichia coli. By measuring phage yield and bacterial growth while changing the growth medium, pH and dissolved oxygen concentration, we found that the optimal conditions for phage yield were NZY medium with pH maintained at 7.4, the dO(2) held at 100% and agitation at 800 rpm. These computer-controlled fermentations result in a minimum of a tenfold higher filamentous phage production compared to standard shake flask conditions.
Assuntos
Reatores Biológicos/virologia , Inovirus/isolamento & purificação , Inovirus/fisiologia , Virologia/métodos , Engenharia Biomédica , Reatores Biológicos/microbiologia , Computadores , Meios de Cultura , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Fermentação , Engenharia Genética , Concentração de Íons de Hidrogênio , Inovirus/genética , Cinética , Biblioteca de PeptídeosRESUMO
Active filamentous phage particles were isolated from the deep-sea bacterium Shewanella piezotolerans WP3. A putative single-stranded DNA binding protein of the phage was found to be overexpressed at 4 degrees C compared to its expression at 25 degrees C by two-dimensional polyacrylamide gel electrophoresis. Reverse transcription quantitative PCR further revealed that the key genes of the SW1 phage were significantly induced at low temperature.